Cotton variety 09r999b2r2

ABSTRACT

The invention relates to the novel cotton variety designated 09R999B2R2. Provided by the invention are the seeds, plants, plant parts and derivatives of the cotton variety 09R999B2R2. Also provided by the invention are tissue cultures of the cotton variety 09R999B2R2 and the plants regenerated therefrom. Still further provided by the invention are methods for producing cotton plants by crossing the cotton variety 09R999B2R2 with itself or another cotton variety and plants produced by such method.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of cotton breeding.In particular, the invention relates to the novel cotton variety09R999B2R2.

2. Description of Related Art

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety animproved combination of desirable traits from the parental germplasm.These important traits may include resistance to diseases and insects,tolerance to drought and heat, tolerance to herbicides, improvements infiber traits and numerous other agronomic traits that may be desirableto the farmer or end user.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of variety used commercially (e.g., F₁ hybrid variety, purelinevariety, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection andbackcrossing.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable variety. This approach hasbeen used extensively for breeding disease-resistant plant varieties.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulvarieties produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for generally three or more years. The best lines arecandidates for new commercial varieties. Those still deficient in a fewtraits may be used as parents to produce new populations for furtherselection.

These processes, which lead to the final step of marketing anddistribution, may take as much as eight to 12 years from the time thefirst cross is made. Therefore, development of new varieties is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to one or more widely grownstandard varieties. Single observations are generally inconclusive,while replicated observations provide a better estimate of geneticworth.

The goal of plant breeding is to develop new, unique and superior cottonvarieties. The breeder initially selects and crosses two or moreparental lines, followed by repeated selfing and selection, producingmany new genetic combinations. Each year, the plant breeder selects thegermplasm to advance to the next generation. This germplasm is grownunder unique and different geographical, climatic and soil conditions,and further selections are then made, during and at the end of thegrowing season. The varieties which are developed are unpredictable.This unpredictability is because the breeder's selection occurs inunique environments, with no control at the DNA level (usingconventional breeding procedures), and with millions of differentpossible genetic combinations being generated. A breeder of ordinaryskill in the art cannot predict the final resulting lines he develops,except possibly in a very gross and general fashion. The same breedercannot produce the same variety twice by using the exact same originalparents and the same selection techniques. This unpredictability resultsin the expenditure of large amounts of research monies to developsuperior new cotton varieties.

Pureline cultivars, such as generally used in cotton and many othercrops, are commonly bred by hybridization of two or more parentsfollowed by selection. The complexity of inheritance, the breedingobjectives and the available resources influence the breeding method.The development of new varieties requires development and selection, thecrossing of varieties and selection of progeny from superior crosses.

Pedigree breeding and recurrent selection breeding methods are used todevelop varieties from breeding populations. Breeding programs combinedesirable traits from two or more varieties or various broad-basedsources into breeding pools from which varieties are developed byselfing and selection of desired phenotypes. The new varieties areevaluated to determine which have commercial potential.

Pedigree breeding is commonly used for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce an F₁. An F₂ population isproduced by selfing one or several F₁ plants. Selection of the bestindividuals may begin in the F₂ population or later depending uponobjectives of the breeder; then, beginning in the F₃, the bestindividuals in the best families can be selected. Replicated testing offamilies can begin in the F₃ or F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are typically tested forpotential release as new varieties.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

The modified single seed descent procedures involve harvesting multipleseed (i.e., a single lock or a simple boll) from each plant in apopulation and combining them to form a bulk. Part of the bulk is usedto plant the next generation and part is put in reserve. This procedurehas been used to save labor at harvest and to maintain adequate seedquantities of the population. The multiple-seed procedure may be used tosave labor. It is considerably faster to gin bolls with a machine thanto remove one seed by hand for the single-seed procedure. Themultiple-seed procedure also makes it possible to plant the same numberof seeds of a population each generation of inbreeding. Enough seeds areharvested to make up for those plants that did not germinate or produceseed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, In: Principles of plant breeding, John Wiley &Sons, NY, University of California, Davis, Calif., 50-98, 1960;Simmonds, In: Principles of crop improvement, Longman, Inc., NY,369-399, 1979; Sneep and Hendriksen, In: Plant breeding perspectives,Wageningen (Ed), Center for Agricultural Publishing and Documentation,1979; Fehr, In: Principles of variety development, Theory and Technique(Vol 1) and Crop Species Soybean (Vol 2), Iowa State Univ., MacmillianPub. Co., NY, 360-376, 1987; Fehr, In: Soybeans: Improvement, Productionand Uses,” 2d Ed., Manograph 16:249, 1987). Additionally, with any ofthe methods disclosed above, mutagenesis can be utilized to increase thediversity of the gene pool that is available in the breeding program.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current varieties. In addition toshowing superior performance, there must be a demand for a new varietythat is compatible with industry standards or which creates a newmarket. The introduction of a new variety will incur additional costs tothe seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new variety should take into consideration research and developmentcosts as well as technical superiority of the final variety. Forseed-propagated varieties, it must be feasible to produce seed easilyand economically.

The two cotton species commercially grown in the United States areGossypium hirsutum, commonly known as short staple or upland cotton andGossypium barbadense, commonly known as extra long staple (ELS) or, inthe United States, as Pima cotton. Upland cotton fiber is used in a widearray of coarser spin count products. Pima cotton is used in finer spincount yarns (50-80) which are primarily used in more expensive garments.Other properties of Pima cotton are critical because of fiber end use.

Cotton is an important and valuable field crop. Thus, a continuing goalof plant breeders is to develop stable, high yielding cotton varietiesthat are agronomically sound. The reasons for this goal are obviously tomaximize the amount and quality of the fiber produced on the land usedand to supply fiber, oil and food for animals and humans. To accomplishthis goal, the cotton breeder must select and develop plants that havethe traits that result in superior cultivars.

The goal of a commercial cotton breeding program is to develop new,unique and superior cotton varieties. In cotton, important traitsinclude higher fiber (lint) yield, earlier maturity, improved fiberquality, resistance to diseases and insects, tolerance to drought andheat, and improved agronomic traits. The breeder initially selects andcrosses two or more parental lines, followed by generation advancementand selection, thus producing many new genetic combinations. The breedercan theoretically generate billions of different genetic combinationsvia this procedure.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to seed of the cottonvariety 09R999B2R2. The invention also relates to plants produced bygrowing the seed of the cotton variety 09R999B2R2, as well as thederivatives of such plants. As used herein, the term “plant” includesplant cells, plant protoplasts, plant cells of a tissue culture fromwhich cotton plants can be regenerated, plant calli, plant clumps, andplant cells that are intact in plants or parts of plants, such aspollen, flowers, seeds, bolls, leaves, stems, and the like.

Another aspect of the invention relates to a tissue culture ofregenerable cells of the cotton variety 09R999B2R2, as well as plantsregenerated therefrom, wherein the regenerated cotton plant expressesall the physiological and morphological characteristics of a plant grownfrom the cotton seed designated 09R999B2R2.

Yet another aspect of the current invention is a cotton plant of thecotton variety 09R999B2R2 comprising at least a first transgene, whereinthe cotton plant is otherwise capable of expressing all thephysiological and morphological characteristics of the cotton variety09R999B2R2. In particular embodiments of the invention, a plant isprovided that comprises a single locus conversion. A single locusconversion may comprise a transgenic gene which has been introduced bygenetic transformation into the cotton variety 09R999B2R2 or aprogenitor thereof. A transgenic or non-transgenic single locusconversion can also be introduced by backcrossing, as is well known inthe art. In certain embodiments of the invention, the single locusconversion may comprise a dominant or recessive allele. The locusconversion may confer potentially any desired trait upon the plant asdescribed herein.

Still yet another aspect of the invention relates to a first generation(F₁) hybrid cotton seed produced by crossing a plant of the cottonvariety 09R999B2R2 to a second cotton plant. Also included in theinvention are the F₁ hybrid cotton plants grown from the hybrid seedproduced by crossing the cotton variety 09R999B2R2 to a second cottonplant. Still further included in the invention are the seeds of an F₁hybrid plant produced with the cotton variety 09R999B2R2 as one parent,the second generation (F₂) hybrid cotton plant grown from the seed ofthe F₁ hybrid plant, and the seeds of the F₂ hybrid plant.

Still yet another aspect of the invention is a method of producingcotton seeds comprising crossing a plant of the cotton variety09R999B2R2 to any second cotton plant, including itself or another plantof the variety 09R999B2R2. In particular embodiments of the invention,the method of crossing comprises the steps of a) planting seeds of thecotton variety 09R999B2R2; b) cultivating cotton plants resulting fromsaid seeds until said plants bear flowers; c) allowing fertilization ofthe flowers of said plants; and, d) harvesting seeds produced from saidplants.

Still yet another aspect of the invention is a method of producinghybrid cotton seeds comprising crossing the cotton variety 09R999B2R2 toa second, distinct cotton plant which is nonisogenic to the cottonvariety 09R999B2R2. In particular embodiments of the invention, thecrossing comprises the steps of a) planting seeds of cotton variety09R999B2R2 and a second, distinct cotton plant, b) cultivating thecotton plants grown from the seeds until the plants bear flowers; c)cross pollinating a flower on one of the two plants with the pollen ofthe other plant, and d) harvesting the seeds resulting from the crosspollinating.

Still yet another aspect of the invention is a method for developing acotton plant in a cotton breeding program comprising: obtaining a cottonplant, or its parts, of the variety 09R999B2R2; and b) employing saidplant or parts as a source of breeding material using plant breedingtechniques. In the method, the plant breeding techniques may be selectedfrom the group consisting of recurrent selection, mass selection, bulkselection, backcrossing, pedigree breeding, genetic marker-assistedselection and genetic transformation. In certain embodiments of theinvention, the cotton plant of variety 09R999B2R2 is used as the male orfemale parent.

Still yet another aspect of the invention is a method of producing acotton plant derived from the cotton variety 09R999B2R2, the methodcomprising the steps of: (a) preparing a progeny plant derived fromcotton variety 09R999B2R2 by crossing a plant of the cotton variety09R999B2R2 with a second cotton plant; and (b) crossing the progenyplant with itself or a second plant to produce a progeny plant of asubsequent generation which is derived from a plant of the cottonvariety 09R999B2R2. In one embodiment of the invention, the methodfurther comprises: (c) crossing the progeny plant of a subsequentgeneration with itself or a second plant; and (d) repeating steps (b)and (c) for at least 2-10 additional generations to produce an inbredcotton plant derived from the cotton variety 09R999B2R2. Also providedby the invention is a plant produced by this and the other methods ofthe invention. Plant variety 09R999B2R2-derived plants produced by thisand the other methods of the invention described herein may, in certainembodiments of the invention, be further defined as comprising thetraits of plant variety 09R999B2R2 given in Table 1.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides, in one aspect, methods and composition relatingto plants, seeds and derivatives of the cotton variety 09R999B2R2. Thecotton variety 09R999B2R2 has been judged to be uniform for breedingpurposes and testing. The variety can be reproduced by planting andgrowing seeds of the variety under self-pollinating or sib-pollinatingconditions, as is known to those of skill in the agricultural arts.Variety 09R999B2R2 shows no variants other than what would normally beexpected due to environment or that would occur for almost anycharacteristic during the course of repeated sexual reproduction.

Variety 09R999B2R2 is an insect-protected and herbicide-tolerant cottonvariety, as a result of containing event MON 15985 and event MON 88913.Event MON 15985 produces the Cry1Ac and Cry2Ab proteins, which areBacillus thuringiensis (subsp. kurstaki) proteins that protect plantsfrom feeding damage caused by lepidopteran pests. MON 88913 produces a5-enolpyruvylshikimate-3-phosphate synthase protein from Agrobacteriumsp. strain CP4 (CP4 EPSPS), which confers tolerance to glyphosate, theactive ingredient in the Roundup® family of agricultural herbicides.

09R999B2R2 is a bulk of three lines (08Y599B2R2, 08Y604B2R2, and08Y607B2R2) developed through backcross breeding using 98L018 as therecurrent parent. The F₁, BC₁, and BC₂ crosses were made in MonsantoCompany's greenhouse in Scott, Miss. The BC₂F₁ plants were grown andselfed in a field near Canas, Costa Rica. The BC₂F₂ plants were grownand selfed in the field near Canas, Costa Rica. Heterozygous MBC₂F₃plants were grown and selfed in the field near Canas, Costa Rica. Plantselection 98L018 [531][15985][88913]-07MBC₂F₄-10391, -10463 and -10559were found to be homozygous for the gene insertions MON 513, 15985, and88913 and each was advanced to a progeny row.

98L018[531][15985][88913]-07MBC₂F₄-10391, -10463 and -10559 were eachplanted in progeny rows in Scott, Miss. Progeny rows were evaluated forgene purity, homozygosity, and trueness to type of the recurrent parent98L018. 98L018[531][15985][88913]-07MBC₂F₄-10391, -10463 and -10559 wereeach selected for increase in Scott, Miss. in May 2007 and given theexperimental names 08Y599B2R2, 08Y604B2R2 and 08Y607B2R2, respectively.Selection was based on visual appearance and acceptable fiberproperties. 08Y599B2R2, 08Y604B2R2, and 08Y607B2R2 were each sent towinter nursery in Costa Rica for increase during the 2007-08 season. In2008, each was increased in Scott, Miss. and Eloy, Ariz. 08Y599B2R2,08Y604B2R2, and 08Y607B2R2 were each sent to winter nursery in CostaRica for increase during the 2008-09 season. The three lines wereevaluated across the cotton belt in replicated research plots in 2008 inthe Monsanto Advanced Testing Program. 08Y599B2R2, 08Y604B2R2, and08Y607B2R2 were very similar phenotypically and for fiber qualities.They were bulked and given the pre-commercial name 09R999B2R2 in 2009.

The results of an objective description of the variety are presentedbelow, in Table 1. Those of skill in the art will recognize that theseare typical values that may vary due to environment and that othervalues that are substantially equivalent are within the scope of theinvention.

TABLE 1 Phenotypic Description of Variety 09R999B2R2 Species: Gossypiumhirsutum L Areas of Adaptation: Eastern Delta Central Plains ArizonaGeneral: Plant Habit: Intermediate Foliage: Intermediate FruitingBranch: Normal Leaf Color: Light green Maturity: % open bolls on ratingdate: 64 Plant: Cm to 1^(st) Fruiting Branch 7 (from cotyledonary node):No. of Nodes to 1^(st) Fruiting Branch 5 (excluding cotyledonary node):Mature Plant Height 104 cm (41 inches) (from cotyledonary node toterminal): Leaf (Upper most, fully expanded leaf): Type: NormalPubsescence: Sparse Nectarines: Present Stem: Stem Pubescence:Intermediate Glands: Leaf: Normal Stem: Normal Calyx lobe (normal isabsent): Normal Flower: Petals: Cream Pollen: Cream Petal Spot: AbsentSeed: Seed Index (g/100 seeds, fuzzy basis): 10.0 Lint Index (g lint/100seeds): 7.0 Boll: Lint percent, picked: 44.0 Number of seeds per boll:26 Number of locules per boll: 4-5 Boll Type: Open Fiber Properties:Method (HVI or other): HVI Length (inches, 2.5% SL): 1.17 Uniformity(%): 85.0 Strength, T1 (g/tex): 30.7 Elongation, E1 (%): 7.4 Micronaire:5.1 Nematodes, Insects and Pests: Bollworm: Resistant Pink bollworm:Resistant Tobacco bud worm: Resistant These are typical values. Valuesmay vary due to environment. Other values that are substantiallyequivalent are within the scope of the invention.

The performance characteristics of cotton variety 09R999B2R2 were alsoanalyzed and comparisons were made with competing varieties. The resultsof the analysis are presented below, in Table 2.

TABLE 2 Performance Data for Variety 09R999B2R2 Variety YLD_BE TSCYD LPMIC LNTH UNIF STR EL FMATR DP 0949B2RF 1388 3314 43.0 4.92 1.15 83.730.0 7.4 86.9 08T737B2R2 1379 3373 42.3 4.67 1.19 83.9 31.8 6.6 87.009R999B2R2 1377 3244 44.0 4.75 1.16 84.3 29.3 7.9 86.1 09R605B2R2 13743298 43.7 4.70 1.17 84.6 29.1 7.9 86.0 09R573B2R2 1354 3196 44.1 4.681.17 84.5 29.6 7.8 86.0 ST5458B2RF 1344 3426 40.8 5.09 1.14 83.3 30.46.5 87.9 DP 0935B2RF 1308 3275 42.4 4.90 1.13 83.3 29.2 7.3 86.8PHY485WRF 1280 3300 40.4 4.84 1.14 84.5 30.9 8.3 86.1 DP164B2RF 12243227 39.4 4.73 1.18 83.9 30.2 6.4 87.1 FM9180B2RF 1169 3151 39.2 4.581.17 84.2 30.8 6.5 86.7 Overall Mean 1318 3224 42.7 4.77 1.16 84.2 30.07.6 86.4 Non-Check 1316 3203 42.9 4.75 1.17 84.3 30.1 7.6 86.3 MeanCheck Mean 1324 3279 42.1 4.80 1.16 84.1 29.9 7.4 86.6 # Locs 30 33 3018 18 18 18 18 17 # Reps 89 94 70 45 45 45 45 45 43 CV 12.1 13.0 2.0 3.61.7 0.7 2.8 5.3 0.6 LSD(.05) 81 203 0.4 0.11 0.01 0.4 0.5 0.3 0.3F-Statistic 6.8 2.2 93.8 21.9 22.6 8.8 16.8 40.3 27.7 P-Value 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 Variety SFC BOP BLSZ FOR SOR OVR PHTLFHAR ST DP 0949B2RF 8.5 66 3.9 2 5 5 40 7 6 08T737B2R2 8.3 60 3.8 2 4 543 5 6 09R999B2R2 8.1 64 4.1 2 4 4 41 4 6 09R605B2R2 8.1 67 3.9 2 4 5 394 5 09R573B2R2 8.1 61 4.2 2 4 5 41 5 5 ST5458B2RF 8.9 65 4.4 2 4 5 39 66 DP 0935B2RF 8.7 60 4.3 2 3 6 41 5 4 PHY485WRF 7.7 66 3.6 3 5 5 41 7 6DP164B2RF 8.4 62 3.8 2 4 5 41 5 6 FM9180B2RF 7.9 66 4.2 1 3 6 37 5 4Overall Mean 8.2 63 4.0 2 4 5 41 5 6 Non-Check 8.1 63 4.0 2 4 5 41 5 6Mean Check Mean 8.2 64 4.1 2 4 5 40 5 5 # Locs 18 16 11 7 10 13 12 2 9 #Reps 45 41 27 20 27 35 27 2 25 CV 6.5 11.1 9.2 24.5 14.3 15.9 8.2 10.110.6 LSD(.05) 0.3 5 0.3 1 1 1 3 1 1 F-Statistic 5.9 2.0 3.2 3.3 6.1 2.12.3 8.6 9.7 P-Value 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

I. BREEDING COTTON VARIETY 09R999B2R2

One aspect of the current invention concerns methods for crossing thecotton variety 09R999B2R2 with itself or a second plant and the seedsand plants produced by such methods. These methods can be used forpropagation of the cotton variety 09R999B2R2, or can be used to producehybrid cotton seeds and the plants grown therefrom. A hybrid plant canbe used as a recurrent parent at any given stage in a backcrossingprotocol during the production of a single locus conversion of thecotton variety 09R999B2R2.

The variety of the present invention is well suited to the developmentof new varieties based on the elite nature of the genetic background ofthe variety. In selecting a second plant to cross with 09R999B2R2 forthe purpose of developing novel cotton varieties, it will typically bedesired to choose those plants which themselves exhibit one or moreselected desirable characteristics. Examples of potentially desiredcharacteristics include higher fiber (lint) yield, earlier maturity,improved fiber quality, resistance to diseases and insects, tolerance todrought and heat, and improved agronomic traits.

Any time the cotton variety 09R999B2R2 is crossed with another,different, variety, first generation (F₁) cotton progeny are produced.The hybrid progeny are produced regardless of characteristics of the twovarieties produced. As such, an F₁ hybrid cotton plant may be producedby crossing 09R999B2R2 with any second cotton plant. The second cottonplant may be genetically homogeneous (e.g., inbred) or may itself be ahybrid. Therefore, any F₁ hybrid cotton plant produced by crossingcotton variety 09R999B2R2 with a second cotton plant is a part of thepresent invention.

Cotton plants can be crossed by either natural or mechanical techniques.Natural pollination occurs in cotton either by self pollination ornatural cross pollination, which typically is aided by pollinatingorganisms. In either natural or artificial crosses, flowering andflowering time are important considerations.

The cotton flower is perfect in that the male and female structures arein the same flower. The crossed or hybrid seed can be produced by manualcrosses between selected parents. Floral buds of the parent that is tobe the female can be emasculated prior to the opening of the flower bymanual removal of the male anthers. At flowering, the pollen fromflowers of the parent plants designated as male, can be manually placedon the stigma of the previous emasculated flower. Seed developed fromthe cross is known as first generation (F₁) hybrid seed. Planting ofthis seed produces F₁ hybrid plants of which half their geneticcomponent is from the female parent and half from the male parent.Segregation of genes begins at meiosis thus producing second generation(F₂) seed. Assuming multiple genetic differences between the originalparents, each F₂ seed has a unique combination of genes.

Self-pollination occurs naturally in cotton with no manipulation of theflowers. For the crossing of two cotton plants, it is typicallypreferable to utilize artificial hybridization. In artificialhybridization, the flower used as a female in a cross is manually crosspollinated prior to maturation of pollen from the flower, therebypreventing self fertilization, or alternatively, the male parts of theflower are emasculated using a technique known in the art. Techniquesfor emasculating the male parts of a cotton flower include, for example,physical removal of the male parts, use of a genetic factor conferringmale sterility, and application of a chemical gametocide to the maleparts.

For artificial hybridization employing emasculation, flowers that areexpected to open the following day are selected on the female parent.The buds are swollen and the corolla is just visible through the calyxor has begun to emerge. Usually no more than two buds on a parent plantare prepared, and all self-pollinated flowers or immature buds areremoved with forceps. Special care is required to remove immature budsthat are hidden under the stipules at the leaf axil, and could developinto flowers at a later date. The flower is grasped between the thumband index finger and the location of the stigma determined by examiningthe sepals. The calyx is removed by grasping a sepal with the forceps,pulling it down and around the flower, and repeating the procedure untilthe five sepals are removed. The exposed corolla is removed with care toavoid injuring the stigma. Cross-pollination can then be carried outusing, for example, petri dishes or envelopes in which male flowers havebeen collected. Desiccators containing calcium chloride crystals can beused in some environments to dry male flowers to obtain adequate pollenshed.

Either with or without emasculation of the female flower, handpollination can be carried out by removing the stamens and pistil with aforceps from a flower of the male parent and gently brushing the anthersagainst the stigma of the female flower. Access to the stamens can beachieved by removing the front sepal and keel petals, or piercing thekeel with closed forceps and allowing them to open to push the petalsaway. Brushing the anthers on the stigma causes them to rupture, and thehighest percentage of successful crosses is obtained when pollen isclearly visible on the stigma. Pollen shed can be checked by tapping theanthers before brushing the stigma. Several male flowers may have to beused to obtain suitable pollen shed when conditions are unfavorable, orthe same male may be used to pollinate several flowers with good pollenshed.

Cross-pollination is more common within rows than between adjacent rows;therefore, it may be preferable to grow populations with genetic malesterility on a square grid to create rows in all directions. Forexample, single-plant hills on 50-cm centers may be used, withsubdivision of the area into blocks of an equal number of hills forharvest from bulks of an equal amount of seed from male-sterile plantsin each block to enhance random pollination.

II. IMPROVEMENT OF COTTON VARIETIES

In certain further aspects, the invention provides plants modified toinclude at least a first desired trait. Such plants may, in oneembodiment, be developed by a plant breeding technique calledbackcrossing, wherein essentially all of the desired morphological andphysiological characteristics of a variety are recovered in addition toa genetic locus transferred into the hybrid via the backcrossingtechnique. The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to a starting variety into whichintroduction of the desired trait is being carried out. The parentalplant which contributes the locus or loci for the desired trait istermed the nonrecurrent or donor parent. This terminology refers to thefact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur.

The parental cotton plant to which the locus or loci from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol (Poehlman andSleper, In: Breeding Field Crops, Iowa State University Press, Ames,1995; Sprague and Dudley, In: Corn and Improvement, 3rd ed., 1988; Fehr,In: Principles of variety development, Theory and Technique (Vol 1) andCrop Species Soybean (Vol 2), Iowa State Univ., Macmillian Pub. Co., NY,360-376, 1987b; Fehr, In: Soybeans: Improvement, Production and Uses,”2d Ed., Manograph 16:249, 1987). In a typical backcross protocol, theoriginal line of interest (recurrent parent) is crossed to a secondvariety (nonrecurrent parent) that carries the genetic locus to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a cottonplant is obtained wherein essentially all of the desired morphologicaland physiological characteristics of the recurrent parent are recoveredin the converted plant, in addition to the transferred locus from thenonrecurrent parent.

The backcross process may be accelerated by the use of genetic markers,such as Simple Sequence Length Polymorphisms (SSLPs) (Williams et al.,Nucleic Acids Res., 18:6531-6535, 1990), Randomly Amplified PolymorphicDNAs (RAPDs), DNA Amplification Fingerprinting (DAF), SequenceCharacterized Amplified Regions (SCARs), Arbitrary Primed PolymeraseChain Reaction (AP-PCR), Amplified Fragment Length Polymorphisms (AFLPs)(EP 534 858, specifically incorporated herein by reference in itsentirety), and Single Nucleotide Polymorphisms (SNPs) (Wang et al.,Science, 280:1077-1082, 1998) to identify plants with the greatestgenetic complement from the recurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto add or substitute one or more new traits in a variety. To accomplishthis, a genetic locus of the recurrent parent is modified or substitutedwith the desired locus from the nonrecurrent parent, while retainingessentially all of the rest of the desired genetic, and therefore thedesired physiological and morphological constitution of the originalplant. The choice of the particular nonrecurrent parent will depend onthe purpose of the backcross; one of the major purposes is to add somecommercially desirable, agronomically important trait to the plant. Theexact backcrossing protocol will depend on the characteristic or traitbeing altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many traits have been identified that are not regularly selected for inthe development of a new variety but that can be improved bybackcrossing techniques. A genetic locus conferring the traits may ormay not be transgenic. Examples of such traits known to those of skillin the art include, but are not limited to, male sterility, herbicidetolerance, resistance for bacterial, fungal, or viral disease, insect ornematode resistance, male sterility, ease of transformation, resistanceto abiotic stresses and improved fiber characteristics. These genes aregenerally inherited through the nucleus, but may be inherited throughthe cytoplasm.

Direct selection may be applied where a genetic locus acts as a dominanttrait. An example of a dominant trait is the herbicide tolerance trait.For this selection process, the progeny of the initial cross are sprayedwith the herbicide prior to the backcrossing. The spraying eliminatesany plants which do not have the desired herbicide tolerancecharacteristic, and only those plants which have the herbicide tolerancegene are used in the subsequent backcross. This process is then repeatedfor all additional backcross generations.

Many useful traits are those which are introduced by genetictransformation techniques. Methods for the genetic transformation ofcotton are known to those of skill in the art, (see, e.g. Firoozabady etal., Plant Mol. Biol., 10:105-116, 1987). For example, broadlyapplicable plant transformation methods which have been describedinclude Agrobacterium-mediated transformation, microprojectilebombardment, electroporation, and direct DNA uptake by protoplasts.

Agrobacterium-mediated transfer is a widely applicable system forintroducing gene loci into plant cells, including cotton. An advantageof the technique is that DNA can be introduced into whole plant tissues,thereby bypassing the need for regeneration of an intact plant from aprotoplast. Modern Agrobacterium transformation vectors are capable ofreplication in E. coli as well as Agrobacterium, allowing for convenientmanipulations (Klee et al., Bio. Tech., 3(7):637-642, 1985). Moreover,recent technological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described haveconvenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes. Additionally, Agrobacterium containing both armed anddisarmed Ti genes can be used for transformation.

In those plant strains where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene locus transfer. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (Fraley et al., Bio. Tech., 3(7):629-635, 1985; U.S.Pat. No. 5,563,055). One efficient means for transformation of cotton inparticular is transformation and regeneration of cotton hypocotylexplants following inoculation with Agrobacterium tumefaciens fromprimary callus development, embryogenesis, embryogenic callusidentification, transgenic cotton shoot production and the developmentof transgenic plants, as is known in the art.

To effect transformation by electroporation, for example, one may employeither friable tissues, such as a suspension culture of cells orembryogenic callus or alternatively one may transform immature embryosor other organized tissue directly. In this technique, one wouldpartially degrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wound tissues ina controlled manner. Protoplasts may also be employed forelectroporation transformation of plants (Bates, Mol. Biotechnol.,2(2):135-145, 1994; Lazzeri, Methods Mol. Biol., 49:95-106, 1995). Forexample, the generation of transgenic cotyledon-derived protoplasts wasdescribed by Dhir and Widholm in Intl. Patent Appl. Publ. No. WO92/17598, the disclosure of which is specifically incorporated herein byreference. When protoplasts are used, transformation can also beachieved using methods based on calcium phosphate precipitation,polyethylene glycol treatment, and combinations of these treatments(see, e.g., Potrykus et al., Mol. Gen. Genet., 199(2):169-177, 1985;Omirulleh et al., Plant Mol. Biol., 21(3):415-428, 1993; Fromm et al.,Nature, 319(6056):791-793, 1986; Uchimiya et al., Mol. Gen. Genet.,204(2):204-207, 1986; Marcotte and Bayley, Nature, 335(6189):454-457,1988).

Microprojectile bombardment is another efficient method for deliveringtransforming DNA segments to plant cells. In this method, particles arecoated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. For the bombardment, cells in suspensionare concentrated on filters or solid culture medium. Alternatively,immature embryos or other target cells may be arranged on solid culturemedium. The cells to be bombarded are positioned at an appropriatedistance below the macroprojectile stopping plate.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. The application ofmicroprojectile bombardment for the transformation of cotton isdescribed, for example, in Rajasekaran et al., Mol. Breed., 2:307-319,1996. An illustrative embodiment of a method for microprojectilebombardment is the Biolistics Particle Delivery System, which can beused to propel particles coated with DNA or cells through a screen, suchas a stainless steel or Nytex screen, onto a surface covered with targetcells. The screen disperses the particles so that they are not deliveredto the recipient cells in large aggregates. It is believed that a screenintervening between the projectile apparatus and the cells to bebombarded reduces the size of projectiles aggregate and may contributeto a higher frequency of transformation by reducing the damage inflictedon the recipient cells by projectiles that are too large.

It is understood to those of skill in the art that a locus of transgenicorigin need not be directly transformed into a plant, as techniques forthe production of stably transformed cotton plants that pass single locito progeny by Mendelian inheritance is well known in the art. Suchsingle loci may therefore be passed from parent plant to progeny plantsby standard plant breeding techniques that are well known in the art.Non-limiting examples of traits that may be introduced directly or bybackcrossing are presented below.

A. Male Sterility

Male sterility genes can increase the efficiency with which hybrids aremade, in that they eliminate the need to physically emasculate the plantused as a female in a given cross. Where one desires to employmale-sterility systems, it may be beneficial to also utilize one or moremale-fertility restorer genes. For example, where cytoplasmic malesterility (CMS) is used, hybrid crossing requires three inbred lines:(1) a cytoplasmically male-sterile line having a CMS cytoplasm; (2) afertile inbred with normal cytoplasm, which is isogenic with the CMSline for nuclear genes (“maintainer line”); and (3) a distinct, fertileinbred with normal cytoplasm, carrying a fertility restoring gene(“restorer” line). The CMS line is propagated by pollination with themaintainer line, with all of the progeny being male sterile, as the CMScytoplasm is derived from the female parent. These male sterile plantscan then be efficiently employed as the female parent in hybrid crosseswith the restorer line, without the need for physical emasculation ofthe male reproductive parts of the female parent.

The presence of a male-fertility restorer gene results in the productionof fully fertile F₁ hybrid progeny. If no restorer gene is present inthe male parent, male-sterile hybrids are obtained. Examples ofmale-sterility genes and corresponding restorers which could be employedwith the plants of the invention are well known to those of skill in theart of plant breeding. Examples of such genes include CMS-D2-2, CMS-hir,CMS-D8, CMS-D4, and CMS-C1. Fertility can be restored to CMS-D2-2 by theD2 restorer in which the restorer factor(s) was introduced from thegenome of G. harknessii Brandegee (D2-2). Microsporogenesis in both CMSsystems aborts during the premeiotic stage. One dominant restorer genefrom the D8 restorer was identified to restore fertility of CMS-D8. TheD2 restorer for CMS-D2-2 also restores the fertility of CMS-D8, CMS-hir,and CMS-C1.

B. Herbicide Tolerance

Numerous herbicide tolerance genes are known and may be employed withthe plants of the invention. An example is a gene conferring toleranceto a herbicide that inhibits the growing point or meristem, such asimidazalinone or sulfonylurea. Exemplary genes in this category code formutant ALS and AHAS enzymes as described, for example, by Lee et al.,EMBO J., 7:1241, 1988; Gleen et al., Plant Molec. Biology, 18:1185-1187,1992; Miki et al., Theor. App. Genet., 80:449, 1990.

Tolerance genes for glyphosate (tolerance conferred by mutant5-enolpyruvl-3 phosphikimate synthase (EPSPS and aroA genes) andhygromycin B phosphotransferase) and to other phosphono compounds suchas glufosinate (phosphinothricin acetyl transferase (PAT) andStreptomyces hygroscopicus phosphinothricin-acetyl transferase (bar)genes) may also be used. For example, U.S. Pat. No. 4,940,835 to Shah,et al., discloses the nucleotide sequence of a form of EPSPS which canconfer glyphosate tolerance. A DNA molecule encoding a mutant aroA genecan be obtained under ATCC accession number 39256, and the nucleotidesequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 toComai. A hygromycin B phosphotransferase gene from E. coli which confersresistance to glyphosate in tobacco callus and plants is described inPenaloza-Vazquez et al. (Plant Cell Reports, 14:482-487, 1995). Europeanpatent application No. 0 333 033 to Kumada et al., and U.S. Pat. No.4,975,374 to Goodman et al., disclose nucleotide sequences of glutaminesynthetase genes which confer tolerance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyltransferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al. DeGreef et al.,(Bio/Technology, 7:61, 1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. Exemplary genes conferring tolerance toherbicidal phenoxy propionic acids and cycloshexones, such as sethoxydimand haloxyfop are the Acct-S1, Accl-S2 and Acct-S3 genes described byMarshall et al., (Theor. App. Genet., 83:435, 1992). U.S. PatentApplication No: 20030135879 describes isolation of a gene for dicambamonooxygenase (DMO) from Psueodmonas maltophilia which is involved inthe conversion of a herbicidal form of the herbicide dicamba to anon-toxic 3,6-dichlorosalicylic acid and thus may be used for producingplants tolerant to this herbicide.

Genes are also known conferring tolerance to a herbicide that inhibitsphotosynthesis, such as triazine (psbA and gs+ genes) and benzonitrile(nitrilase gene). Przibilla et al., (Plant Cell, 3:169, 1991), describethe transformation of Chlamydomonas with plasmids encoding mutant psbAgenes. Nucleotide sequences for nitrilase genes are disclosed in U.S.Pat. No. 4,810,648 to Stalker, and DNA molecules containing these genesare available under ATCC Accession Nos. 53435, 67441, and 67442. Cloningand expression of DNA coding for a glutathione S-transferase isdescribed by Hayes et al., (Biochem. J. 285:173, 1992).Protoporphyrinogen oxidase (PPO) is the target of the PPO-inhibitorclass of herbicides; a PPO-inhibitor resistant PPO gene was recentlyidentified in Amaranthus tuberculatus (Patzoldt et al., PNAS,103(33):12329-2334, 2006). The herbicide methyl viologen inhibits CO₂assimilation. Foyer et al. (Plant Physiol, 109:1047-1057, 1995) describea plant overexpressing glutathione reductase (GR) which is resistant tomethyl viologen treatment.

Siminszky (Phytochemistry Reviews, 5:445-458, 2006) describes plantcytochrome P450-mediated detoxification of multiple, chemicallyunrelated classes of herbicides.

Other examples of herbicide resistance have been described, forinstance, in U.S. Pat. Nos. 6,803,501; 6,448,476; 6,248,876; 6,225,114;6,107,549; 5,866,775; 5,804,425; 5,633,435; 5,463,175.

C. Disease Resistance

Plant defenses are often activated by specific interaction between theproduct of a disease resistance gene (R) in the plant and the product ofa corresponding avirulence (Avr) gene in the pathogen. A plant line canbe transformed with cloned resistance gene to engineer plants that areresistant to specific pathogen strains. See, for example Jones et al.,Science, 266:789, 1994 (cloning of the tomato Cf-9 gene for resistanceto Cladosporium fulvum); Martin et al., Science, 262:1432, 1993 (tomatoPto gene for resistance to Pseudomonas syringae pv.); Mindrinos et al.,Cell, 78:1089, 1994 (Arabidopsis RPS2 gene for resistance to Pseudomonassyringae). Logemann et al., (Bio/technology, 10:305, 1992), for example,disclose transgenic plants expressing a barley ribosome-inactivatinggene have an increased resistance to fungal disease.

A viral-invasive protein or a complex toxin derived therefrom may alsobe used for viral disease resistance. For example, the accumulation ofviral coat proteins in transformed plant cells imparts resistance toviral infection and/or disease development effected by the virus fromwhich the coat protein gene is derived, as well as by related viruses.See Beachy et al., Ann. Rev. Phytopathol., 28:451, 1990. Coatprotein-mediated resistance has been conferred upon transformed plantsagainst alfalfa mosaic virus, cucumber mosaic virus, tobacco streakvirus, potato virus X, potato virus Y, tobacco etch virus, tobaccorattle virus and tobacco mosaic virus.

A virus-specific antibody may also be used. See, for example,Tavladoraki et al., (Nature, 366:469, 1993), who show that transgenicplants expressing recombinant antibody genes are protected from virusattack. Additional means of inducing whole-plant resistance to apathogen include modulation of the systemic acquired resistance (SAR) orpathogenesis related (PR) genes, for example genes homologous to theArabidopsis thaliana NIM1/NPR1/SAI1, and/or increasing salicylic acidproduction (Ryals et al., Plant Cell, 8:1809-1819, 1996).

Plant defensins may be used to provide resistance to fungal pathogens(Thomma et al., Planta, 216:193-202, 2002).

Nematode resistance has been described, for example, in U.S. Pat. No.6,228,992 and bacterial disease resistance in U.S. Pat. No. 5,516,671.

D. Insect Resistance

One example of an insect resistance gene includes a Bacillusthuringiensis protein, a derivative thereof, or a synthetic polypeptidemodeled thereon. See, for example, Geiser et al., (Gene, 48:109, 1986),who disclose the cloning and nucleotide sequence of a Bt δ-endotoxingene. Moreover, DNA molecules encoding δ-endotoxin genes can bepurchased from the American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.Another example is a lectin. See, for example, Van Damme et al., (PlantMolec. Biol., 24:25, 1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes. A vitamin-bindingprotein may also be used, such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.This application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

Yet another insect resistance gene is an enzyme inhibitor, for example,a protease or proteinase inhibitor or an amylase inhibitor. See, forexample, Abe et al., J. Biol. Chem., 262:16793, 1987 (nucleotidesequence of rice cysteine proteinase inhibitor), Huub et al., PlantMolec. Biol., 21:985, 1993 (nucleotide sequence of cDNA encoding tobaccoproteinase inhibitor I), and Sumitani et al., Biosci. Biotech. Biochem.,57:1243, 1993 (nucleotide sequence of Streptomyces nitrosporeusα-amylase inhibitor).

An insect-specific hormone or pheromone may also be used. See, forexample, Hammock et al., (Nature, 344:458, 1990) disclosing baculovirusexpression of cloned juvenile hormone esterase, an inactivator ofjuvenile hormone, Gade and Goldsworthy (Eds. Physiological System inInsects, Elsevier Academic Press, Burlington, Mass., 2007), describingallostatins and their potential use in pest control; and Palli et al.(Vitam. Horm., 73:59-100, 2005), disclosing use of ecdysteroid andecdysteroid receptor in agriculture. The diuretic hormone receptor (DHR)was identified in Price et al. (Insect Mol. Biol., 13:469-480, 2004) asa candidate target of insecticides.

Still other examples include an insect-specific antibody or animmunotoxin derived therefrom and a developmental-arrestive protein. SeeTaylor et al., (Seventh Int'l Symposium on Molecular Plant-MicrobeInteractions (Edinburgh, Scotland) Abstract #497, 1994), who describedenzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments.

E. Resistance to Abiotic Stresses

Abiotic stress includes dehydration or other osmotic stress, salinity,high or low light intensity, high or low temperatures, submergence,exposure to heavy metals, and oxidative stress.Delta-pyrroline-5-carboxylate synthetase (P5CS) from mothbean has beenused to provide protection against general osmotic stress.Mannitol-1-phosphate dehydrogenase (mt1D) from E. coli has been used toprovide protection against drought and salinity. Choline oxidase (codAfrom Arthrobactor globiformis) can protect against cold and salt. E.coli choline dehydrogenase (betA) provides protection against salt.Additional protection from cold can be provided by omega-3-fatty aciddesaturase (fad7) from Arabidopsis thaliana. Trehalose-6-phosphatesynthase and levan sucrase (SacB) from yeast and Bacillus subtilis,respectively, can provide protection against drought (summarized fromAnnex II Genetic Engineering for Abiotic Stress Tolerance in Plants,Consultative Group On International Agricultural Research TechnicalAdvisory Committee). Overexpression of superoxide dismutase can be usedto protect against superoxides, as described in U.S. Pat. No. 5,538,878to Thomas et al.

F. Modified Fatty Acid, Phytate and Carbohydrate Metabolism

Genes may be used conferring modified fatty acid metabolism. Forexample, stearyl-ACP desaturase genes may be used. See Knutzon et al.,Proc. Natl. Acad. Sci. USA, 89:2624, 1992. Various fatty aciddesaturases have also been described, such as a Saccharomyces cerevisiaeOLE1 gene encoding delta-9 fatty acid desaturase, an enzyme which formsthe monounsaturated palmitoleic (16:1) and oleic (18:1) fatty acids frompalmitoyl (16:0) or stearoyl (18:0) CoA (McDonough et al., J. Biol.Chem., 267(9):5931-5936, 1992); a gene encoding a stearoyl-acyl carrierprotein Δ9 desaturase from castor (Fox et al. Proc. Natl. Acad. Sci.USA, 90(6):2486-2490, 1993); Δ6 and Δ12 desaturases from thecyanobacteria Synechocystis responsible for the conversion of linoleicacid (18:2) to gamma-linolenic acid (18:3 gamma) (Reddy et al. PlantMol. Biol., 22(2):293-300, 1993); a gene from Arabidopsis thaliana thatencodes an omega-3 desaturase (Arondel et al. Science,258(5086):1353-1355, 1992); plant Δ9 desaturases (PCT Application Publ.No. WO 91/13972) and soybean and Brassica Δ15 desaturases (EuropeanPatent Application Publ. No. EP 0616644).

Phytate metabolism may also be modified by introduction of aphytase-encoding gene to enhance breakdown of phytate, adding more freephosphate to the transformed plant. For example, see Van Hartingsveldtet al., (Gene, 127:87, 1993), for a disclosure of the nucleotidesequence of an Aspergillus niger phytase gene. This, for example, couldbe accomplished by cloning and then reintroducing DNA associated withthe single allele which is responsible for mutants characterized by lowlevels of phytic acid. See Raboy et al., (Maydica, 35:383, 1990).

A number of genes are known that may be used to alter carbohydratemetabolism. For example, plants may be transformed with a gene codingfor an enzyme that alters the branching pattern of starch. See Shirozaet al., J. Bacteol., 170:810, 1988 (nucleotide sequence of Streptococcusmutans fructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet.,20:220, 1985 (nucleotide sequence of Bacillus subtilis levansucrasegene), Pen et al., BioTechnology, 10:292, 1992 (production of transgenicplants that express Bacillus licheniformis α-amylase), Elliot et al.,Plant Molec. Biol., 21:515, 1993 (nucleotide sequences of tomatoinvertase genes), Sergaard et al., J. Biol. Chem., 268:22480, 1993(site-directed mutagenesis of barley α-amylase gene), and Fisher et al.,Plant Physiol., 102:1045, 1993 (maize endosperm starch branching enzymeII). The Z10 gene encoding a 10 kD zein storage protein from maize mayalso be used to alter the quantities of 10 kD Zein in the cells relativeto other components (Kirihara et al., Mol. Gen. Genet., 211:477-484,1988).

G. Improved Cotton Fiber Characteristics

Fiber characteristics such as fiber quality of quantity representanother example of a trait that may be modified in cotton varieties. Forexample, U.S. Pat. No. 6,472,588 describes transgenic cotton plantstransformed with a sucrose phosphate synthase nucleic acid to alterfiber characteristics such as strength, length, fiber fineness, fibermaturity ratio, immature fiber content, fiber uniformity, andmicronaire. Cotton plants comprising one or more genes coding for anenzyme selected from the group consisting of endoxyloglucan transferase,catalase and peroxidase for the improvement of cotton fibercharacteristics are also described in U.S. Pat. No. 6,563,022. Cottonmodification using ovary-tissue transcriptional factors preferentiallydirecting gene expression in ovary tissue, particularly in very earlyfruit development, utilized to express genes encoding isopentenyltransferase in cotton ovule tissue and modify the characteristics ofboll set in cotton plants and alter fiber quality characteristicsincluding fiber dimension and strength is discussed in U.S. Pat. No.6,329,570. A gene controlling the fiber formation mechanism in cottonplants is described in U.S. Pat. No. 6,169,174.

Genes involved in lignin biosynthesis are described by Dwivedi et al.,Mol. Biol., 26:61-71, 1994; Tsai et al., Physiol., 107:1459, 1995; U.S.Pat. No. 5,451,514 (claiming the use of cinnamyl alcohol dehydrogenasegene in an antisense orientation such that the endogenous plant cinnamylalcohol dehydrogenase gene is inhibited).

H. Additional Traits

Additional traits can be introduced into the cotton variety of thepresent invention. A non-limiting example of such a trait is a codingsequence which decreases RNA and/or protein levels. The decreased RNAand/or protein levels may be achieved through RNAi methods, such asthose described in U.S. Pat. No. 6,506,559 to Fire and Mellow.

Another trait that may find use with the cotton variety of the inventionis a sequence which allows for site-specific recombination. Examples ofsuch sequences include the FRT sequence, used with the FLP recombinase(Zhu and Sadowski, J. Biol. Chem., 270:23044-23054, 1995); and the LOXsequence, used with CRE recombinase (Sauer, Mol. Cell. Biol.,7:2087-2096, 1987). The recombinase genes can be encoded at any locationwithin the genome of the cotton plant, and are active in the hemizygousstate.

It may also be desirable to make cotton plants more tolerant to or moreeasily transformed with Agrobacterium tumefaciens. Expression of p53 andiap, two baculovirus cell-death suppressor genes, inhibited tissuenecrosis and DNA cleavage. Additional targets can include plant-encodedproteins that interact with the Agrobacterium Vir genes; enzymesinvolved in plant cell wall formation; and histones, histoneacetyltransferases and histone deacetylases (reviewed in Gelvin,Microbiology & Mol. Biol. Reviews, 67:16-37, 2003).

III. TISSUE CULTURES AND IN VITRO REGENERATION OF COTTON PLANTS

A further aspect of the invention relates to tissue cultures of thecotton variety designated 09R999B2R2. As used herein, the term “tissueculture” indicates a composition comprising isolated cells of the sameor a different type or a collection of such cells organized into partsof a plant. Exemplary types of tissue cultures are protoplasts, calliand plant cells that are intact in plants or parts of plants, such asembryos, pollen, flowers, leaves, roots, root tips, anthers, and thelike. In a preferred embodiment, the tissue culture comprises embryos,protoplasts, meristematic cells, pollen, leaves or anthers.

An important ability of a tissue culture is the capability to regeneratefertile plants. This allows, for example, transformation of the tissueculture cells followed by regeneration of transgenic plants. Fortransformation to be efficient and successful, DNA must be introducedinto cells that give rise to plants or germ-line tissue.

Plants typically are regenerated via two distinct processes; shootmorphogenesis and somatic embryogenesis. Shoot morphogenesis is theprocess of shoot meristem organization and development. Shoots grow outfrom a source tissue and are excised and rooted to obtain an intactplant. During somatic embryogenesis, an embryo (similar to the zygoticembryo), containing both shoot and root axes, is formed from somaticplant tissue. An intact plant rather than a rooted shoot results fromthe germination of the somatic embryo.

Shoot morphogenesis and somatic embryogenesis are different processesand the specific route of regeneration is primarily dependent on theexplant source and media used for tissue culture manipulations. Whilethe systems are different, both systems show variety-specific responseswhere some lines are more responsive to tissue culture manipulationsthan others. A line that is highly responsive in shoot morphogenesis maynot generate many somatic embryos. Lines that produce large numbers ofembryos during an induction step may not give rise to rapidly-growingproliferative cultures. Therefore, it may be desired to optimize tissueculture conditions for each cotton line. These optimizations may readilybe carried out by one of skill in the art of tissue culture throughsmall-scale culture studies. In addition to line-specific responses,proliferative cultures can be observed with both shoot morphogenesis andsomatic embryogenesis. Proliferation is beneficial for both systems, asit allows a single, transformed cell to multiply to the point that itwill contribute to germ-line tissue.

Embryogenic cultures can also be used successfully for regeneration,including regeneration of transgenic plants, if the origin of theembryos is recognized and the biological limitations of proliferativeembryogenic cultures are understood. Biological limitations include thedifficulty in developing proliferative embryogenic cultures and reducedfertility problems (culture-induced variation) associated with plantsregenerated from long-term proliferative embryogenic cultures. Some ofthese problems are accentuated in prolonged cultures. The use of morerecently cultured cells may decrease or eliminate such problems.

IV. DEFINITIONS

In the description and tables herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided:

A: When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more.”

Allele: Any of one or more alternative forms of a gene locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny.

Backcrossing can be used to introduce one or more single locusconversions from one genetic background into another.

Crossing: The mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Desired Agronomic Characteristics: Agronomic characteristics (which willvary from crop to crop and plant to plant) such as yield, maturity, pestresistance and lint percent which are desired in a commerciallyacceptable crop or plant. For example, improved agronomiccharacteristics for cotton include yield, maturity, fiber content andfiber qualities.

Diploid: A cell or organism having two sets of chromosomes.

Disease Resistance: The ability of plants to restrict the activities ofa specified pest, such as an insect, fungus, virus, or bacteria.

Disease Tolerance: The ability of plants to endure a specified pest(such as an insect, fungus, virus or bacteria) or an adverseenvironmental condition and still perform and produce in spite of thisdisorder.

Donor Parent: The parent of a variety which contains the gene or traitof interest which is desired to be introduced into a second variety.

ELS: The abbreviation for “Extra Long Staple.” ELS is the groupclassification for cotton in the longest staple length category. As usedin practice and for commerce, ELS denotes varieties belonging to thespecies G. barbadense that have superior fiber qualities, includingclassification in the longest staple length category.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a cytoplasmic or nuclear genetic factor conferring malesterility or a chemical agent.

Essentially all of the physiological and morphological characteristics:A plant having essentially all of the physiological and morphologicalcharacteristics of a designated plant has all of the characteristics ofthe plant that are otherwise present when compared in the sameenvironment, other than an occasional variant trait that might ariseduring backcrossing or direct introduction of a transgene.

F₁ Hybrid: The first generation progeny of the cross of two nonisogenicplants.

Fallout (Fo): As used herein, the term “fallout” refers to the rating ofhow much cotton has fallen on the ground at harvest.

2.5% Fiber Span Length: Refers to the longest 2.5% of a bundle of fibersexpressed in inches as measured by a digital fibergraph.

Fiber Characteristics: Refers to fiber qualities such as strength, fiberlength, micronaire, fiber elongation, uniformity of fiber and amount offiber.

Fiber Elongation: Sometimes referred to as E1, refers to the elongationof the fiber at the point of breakage in the strength determination asmeasured by High Volume Instrumentation (HVI).

2.5% Fiber Span Length: Refers to the longest 2.5% of a bundle of fibersexpressed in inches as measured by a digital fibergraph.

Fiber Span Length: The distance spanned by a specific percentage offibers in a test specimen, where the initial starting point of thescanning in the test is considered 100 percent as measured by a digitalfibergraph.

Fiber Strength: Also referred to as T1, denotes the force required tobreak a bundle of fibers. Fiber strength is expressed in millinewtons(mn) per tex on a stelometer.

Fruiting Nodes: The number of nodes on the main stem from which arisebranches that bear fruit or boll in the first position.

Genotype: The genetic constitution of a cell or organism.

Gin Turnout: Refers to fraction of lint in a machine harvested sample ofseed cotton (lint, seed, and trash).

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Lint Index: The weight of lint per seed in grams.

Lint Percent Refers to the lint (fiber) fraction of seed cotton (lintand seed).

Lint Yield Refers to the measure of the quantity of fiber produced on agiven unit of land. Presented herein in pounds of lint per acre.

Lint/boll: As used herein, the term “lint/boll” is the weight of lintper boll.

Maturity Rating: A visual rating near harvest on the amount of openbolls on the plant. The rating range is from 1 to 5, 1 being early and 5being late.

Micronaire: A measure of the fineness of the fiber. Within a cottoncultivar, micronaire is also a measure of maturity. Micronairedifferences are governed by changes in perimeter or in cell wallthickness, or by changes in both. Within a variety, cotton perimeter isfairly consistent and maturity will cause a change in micronaire.Consequently, micronaire has a high correlation with maturity within avariety of cotton. Maturity is the degree of development of cell wallthickness. Micronaire may not have a good correlation with maturitybetween varieties of cotton having different fiber perimeter. Micronairevalues range from about 2.0 to 6.0:

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Plant Height: The average height in meters of a group of plants.

Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Recurrent Parent: The repeating parent (variety) in a backcross breedingprogram. The recurrent parent is the variety into which a gene or traitis desired to be introduced.

Regeneration: The development of a plant from tissue culture.

Seed/boll: Refers to the number of seeds per boll.

Seedcotton/boll: Refers to the weight of seedcotton per boll.

Seedweight: Refers to the weight of 100 seeds in grams.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant or a plant of the same genotype.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing wherein essentially allof the desired morphological and physiological characteristics of avariety are recovered in addition to the characteristics conferred bythe single locus transferred into the variety via the backcrossingtechnique. A single locus may comprise one gene, or in the case oftransgenic plants, one or more transgenes integrated into the hostgenome at a single site (locus).

Stringout Rating: also sometimes referred to as “Storm Resistance”refers to a visual rating prior to harvest of the relative looseness ofthe seed cotton held in the boll structure on the plant. The ratingvalues are from 1 to 5 (tight to loose in the boll).

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference (e.g., p=0.05) from themean.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a cotton plant by transformation.

Uniformity Ratio: A measure of the relative fiber span length uniformityof a bundle of fibers. The uniformity ratio is determined by dividingthe 50% fiber span length by the 2.5% fiber span length.

Vegetative Nodes: The number of nodes from the cotyledonary node to thefirst fruiting branch on the main stem of the plant.

V. DEPOSIT INFORMATION

Applicant will make a deposit of at least 2500 seeds of cotton variety09R999B2R2 disclosed herein with the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA. Theaccession number for the deposit is ATCC Accession No. PTA-______ andthe date of deposit is ______. Access to this deposit will be availableduring the pendency of the application to the Commissioner of Patentsand Trademarks and persons determined by the Commissioner to be entitledthereto upon request. The deposit will be maintained for a period of 30years, or 5 years after the most recent request, or for the enforceablelife of the patent, whichever is longer, and will be replaced if itbecomes nonviable during that period. Applicant does not waive anyinfringement of rights granted under this patent or under the PlantVariety Protection Act (7 U.S.C. 2321 et seq.).

The references cited herein, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

1. A plant of cotton variety 09R999B2R2, wherein a sample of seed ofsaid variety has been deposited under ATCC Accession No. ______.
 2. Aplant part of the plant of claim 1, wherein the plant part comprises atleast a first cell of said plant.
 3. The plant part of claim 3, furtherdefined as pollen, a meristem or an ovule.
 4. A cell of the plant ofclaim
 1. 5. A tissue culture of regenerable cells comprising at least acell of claim
 4. 6. A seed of cotton variety 09R999B2R2, wherein asample of seed of said variety has been deposited under ATCC AccessionNo. ______.
 7. A cotton plant regenerated from the tissue culture ofclaim 5, wherein the regenerated cotton plant expresses all of thephysiological and morphological characteristics of the cotton variety09R999B2R2.
 8. A method of producing cotton seed, comprising crossingthe plant of claim 1 with itself or a second cotton plant.
 9. The methodof claim 8, defined as comprising crossing the plant of claim 1 with asecond, distinct cotton plant.
 10. An F₁ hybrid cotton seed produced bythe method of claim
 9. 11. An F₁ hybrid cotton plant produced by growingthe seed of claim
 10. 12. A method of producing a cotton plantcomprising an added desired trait, the method comprising introducing atransgene conferring the desired trait into a plant of cotton variety09R999B2R2, wherein a sample of seed of said variety has been depositedunder ATCC Accession No. ______.
 13. The method of claim 12, comprisingintroducing the transgene by backcrossing.
 14. The method of claim 12,comprising introducing the transgene by genetic transformation.
 15. Themethod of claim 12, wherein the desired trait is selected from the groupconsisting of male sterility, herbicide tolerance, insect or pestresistance, disease resistance, modified fatty acid metabolism, modifiedcarbohydrate metabolism and modified cotton fiber characteristics. 16.The method of claim 15, wherein the desired trait is herbicide toleranceand the tolerance is conferred to an herbicide selected from the groupconsisting of glyphosate, sulfonylurea, imidazalinone, dicamba,glufosinate, phenoxy proprionic acid, cyclohexanedione, triazine,benzonitrile and broxynil.
 17. The method of claim 15, wherein thedesired trait is insect resistance and the transgene encodes a Bacillusthuringiensis (Bt) endotoxin.
 18. A cotton plant produced by the methodof claim
 12. 19. A method of introducing a single locus conversion intocotton variety 09R999B2R2 comprising: (a) crossing a plant of variety09R999B2R2 with a second plant to produce cotton seed, wherein thesecond plant comprises a desired single locus and wherein a sample ofseed of said variety has been deposited under ATCC Accession No. ______;(b) growing F1 progeny plants from the seed and selecting at least afirst a first F1 progeny plant that has the single locus to produceselected F1 progeny plants; (c) crossing the selected progeny plantswith at least a first plant of variety 09R999B2R2 to produce backcrossprogeny plants; (d) selecting backcross progeny plants that have thesingle locus and physiological and morphological characteristics ofcotton variety 09R999B2R2 to produce selected backcross progeny plants;and (e) repeating steps (c) and (d) in succession until backcrossprogeny plants are obtained that comprise the single locus and otherwisecomprise essentially all of the physiological and morphologicalcharacteristics of cotton variety 09R999B2R2 when grown in the sameenvironmental conditions.
 20. The method of claim 19, wherein the singlelocus confers a trait selected from the group consisting of malesterility; herbicide tolerance; insect or pest resistance; diseaseresistance; modified fatty acid metabolism; modified carbohydratemetabolism; and modified cotton fiber characteristics.
 21. The method ofclaim 20, wherein the trait is tolerance to an herbicide selected fromthe group consisting of glyphosate, sulfonylurea, imidazalinone,dicamba, glufosinate, phenoxy proprionic acid, cyclohexanedione,triazine, benzonitrile and broxynil.
 22. The method of claim 20, whereinthe trait is insect resistance and the insect resistance is conferred bya transgene encoding a Bacillus thuringiensis (Bt) endotoxin.
 23. Aplant produced by introducing a single locus conversion into cottonvariety 09R999B2R2, or a selfed progeny of said plant that comprises thesingle locus conversion, wherein the single locus was introduced intocotton variety 09R999B2R2 by backcrossing or genetic transformation andwherein representative seed of said cotton variety 09R999B2R2 have beendeposited under ATCC Accession No. ______.
 24. A seed that produces theplant of claim
 23. 25. A method of producing a cotton plant derived fromthe cotton variety 09R999B2R2, the method comprising crossing a plant ofvariety 09R999B2R2 with a second, distinct cotton plant to produce acotton plant derived from the cotton variety 09R999B2R2, wherein asample of seed of said variety has been deposited under ATCC AccessionNo. ______.
 26. The method of claim 25 further comprising: (a) crossingthe plant derived from the cotton variety 09R999B2R2 with itself or adifferent cotton plant to produce a seed of a progeny plant of asubsequent generation; (b) growing a progeny plant of a subsequentgeneration from said seed and crossing the progeny plant of a subsequentgeneration with itself or a second plant; and (c) repeating steps (a)and (b) with sufficient inbreeding until an inbred cotton plant derivedfrom the cotton variety 09R999B2R2 is produced.
 27. The method of claim26, comprising crossing said inbred cotton plant derived from the cottonvariety 09R999B2R2 with a plant of a different genotype to produce aseed of a hybrid cotton plant derived from the cotton variety09R999B2R2.
 28. A commodity plant product comprising at least a firstcell according to claim
 4. 29. The commodity plant product of claim 28,wherein the commodity plant product is lint or cotton seed oil.